Project Summary Cell adhesion molecules are critical for synaptic development in the CNS. They mediate homophilic binding and trans-synaptic interactions, both stabilizing synapses and specifying connectivity. Defects in synaptic adhesion are thought to accompany some neurodevelopmental disorders such as autism and mental retardation. The discovery of the clustered and non-clustered protocadherins (Pcdhs), the largest family of adhesion molecules, generated great interest for their potential role as synaptic specifiers. Our studies of one Pcdh subfamily (the Pcdh-?s) point to a much different role for the Pcdhs in cellular interactions at the synapse. We showed that Pcdh-?s have homophilic binding specificity at the cell surface, consistent with a role in cell- cell interactions, but we also found that both endogenous and expressed Pcdh-?s are mostly found in intracellular compartments rather than the cell surface. This contrasted with the surface distribution of classical cadherins. In further support of a nonconventional role for Pcdhs, we identified specific targeting signals in the cytoplasmic domains of Pcdh-?s that mediate their retention or endocytosis in the endolysosome system. The Pcdh intracellular distribution and the homophilic specificity of their extracellular domains represent a paradox: How can Pcdhs participate in cell-cell interactions if they are not at the surface? There are two possibilities for how Pcdh cell-cell engagement might affect the synapse. The dendrite self-avoidance activity of Pcdh-?s suggests that they might mediate avoidance at synapses that express matching Pcdhs, which would implicate them in synaptic destabilization/pruning during development. Alternatively, they may have a pro-adhesive role at the synapse, resulting in synaptic stabilization and maturation. Given the emerging importance of Pcdhs in synaptic neurodevelopmental disorders such as autism, which has a clear synaptic component, it will be important that we differentiate between the two possibilities. In this proposal we will address for the first time how Pcdhs might affect synaptic development by conducting quantitative serial immuno-electron microscopic analysis of Pcdh synaptic localization and how their trafficking associates with synaptic development and morphology. We will also begin studies of two non-clustered protocadherins, Pcdh-8 and Pcdh-10, which have not yet been characterized at the synaptic level.